Thomas Decoville

Nonneutralizing Antibodies Are Able To Inhibit Human Immunodeficiency Virus Type 1 Replication in Macrophages and Immature Dendritic Cells

ABSTRACT Only five monoclonal antibodies (MAbs) neutralizing a broad range of primary isolates (PI) have been identified up to now. We have found that some MAbs with no neutralizing activities according to the “conventional” neutralization assay, involving phytohemagglutinin-stimulated peripheral blood mononuclear cells as targets, efficiently inhibit the replication of human immunodeficiency virus type 1 (HIV-1) PI in macrophages and immature dendritic cells (iDC). The mechanism of inhibition is distinct from the neutralization of infectivity occurring via Fab fragments and involves the interaction of the F portion with the FcγRs present on macrophages and iDC. We propose that, if such nonneutralizing inhibitory antibodies limit mucosal HIV transmission, they should be induced by vaccination.

Neutralizing antibodies inhibit HIV-1 transfer from primary dendritic cells to autologous CD4 T-lymphocytes

Dendritic cells (DCs) support only low levels of HIV-1 replication, but have been shown to transfer infectious viral particles highly efficiently to neighboring permissive CD4 T lymphocytes. This mode of cell-to-cell HIV-1 spread may be a predominant mode of infection and dissemination. In the present study, we analyzed the kinetics of fusion, replication, and the ability of HIV-1-specific Abs to inhibit HIV-1 transfer from immature DCs to autologous CD4 T lymphocytes. We found that neutralizing mAbs prevented HIV-1 transfer to CD4 T lymphocytes in trans and in cis, whereas nonneutralizing Abs did not. Neutralizing Abs also significantly decreased HIV-1 replication in DCs, even when added 2 hours after HIV-1 infection. Interestingly, a similar inhibition of HIV-1 replication in DCs was detected with some nonneutralizing Abs and was correlated with DC maturation. We suggest that the binding of HIV-1-specific Abs to FcγRs leads to HIV-1 inhibition in DCs by triggering DC maturation. This efficient inhibition of HIV-1 transfer by Abs highlights the importance of inducing HIV-specific Abs by vaccination directly at the mucosal portal of HIV-1 entry to prevent early dissemination after sexual transmission.

Efficient transfer of HIV-1 in trans and in cis from Langerhans dendritic cells and macrophages to autologous T lymphocytes.

Objective:The chronology of HIV infection in mucosal tissue after sexual transmission is unknown. Several potential HIV target cells are present at these sites, including dendritic cells, macrophages, and CD4+ T lymphocytes. Dendritic cells and macrophages are antigen-presenting cells (APCs) and are thus involved in cross-talk with T cells. This close contact may favor efficient HIV-1 transfer to T lymphocytes, resulting in rapid HIV-1 dissemination. Design:We investigated the role of APCs in HIV transfer to T cells by incubating Langerhans cells and interstitial dendritic cells (IDCs) or monocyte-derived macrophages (MDMs) with HIV for 2 h before addition of uninfected autologous CD4+ T lymphocytes. Methods:HIV infection was recorded after different time points. Following staining, the measurement of intracellular p24 in the different cell populations was analyzed by flow cytometry. Results:We showed that Langerhans cells/IDCs and macrophages efficiently transferred HIV to CD4+ T cells. Interestingly, a rapid HIV transfer in trans predominated in MDMs, whereas cis transfer mainly occurred in Langerhans cells/IDC cocultures. Neutralizing antibody 2G12, added to HIV-loaded APCs, efficiently blocked both the trans and the cis infection of T cells. Conclusion:These findings highlight the major contributions of various mucosal cells in HIV dissemination and suggest that HIV hijacks the different properties of APCs to favor its dissemination through the body. They emphasize the role of macrophages in the rapid transmission of HIV to T lymphocytes at mucosal sites, dendritic cells being prone to migration to lymphoid organ for subsequent dissemination by cis transfer.

Dendritic Cell-Lymphocyte Cross Talk Downregulates Host Restriction Factor SAMHD1 and Stimulates HIV-1 Replication in Dendritic Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and simian immunodeficiency virus (SIV) from sooty mangabeys (SIVsm) or from macaques (SIVmac) but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T and B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome under coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC-lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with increased HIV-1 replication in DCs. Moreover, in infected DC-T lymphocyte cocultures, DCs acquired maturation status and secreted type 1 interferon (alpha interferon [IFN-α]). The blockade of DC-lymphocyte cross talk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, cross talk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture. IMPORTANCE SAMHD1 restricts HIV-1 replication in dendritic cells (DCs). Here, we demonstrate that, in a coculture model of DCs and lymphocytes mimicking early mucosal HIV-1 infection, stimulation of HIV-1 replication in DCs is associated with downregulation of SAMHD1 expression and activation of innate immune sensing by DCs. We propose that DC-lymphocyte cross talk occurring in vivo modulates host restriction factor SAMHD1, promoting HIV-1 replication in cellular reservoirs and stimulating immune sensing.

Stimulation of HIV-1 Replication in Immature Dendritic Cells in Contact with Primary CD4 T or B Lymphocytes

ABSTRACT Sexual transmission is the major route of HIV-1 infection worldwide. Dendritic cells (DCs) from the mucosal layers are considered to be the initial targets of HIV-1 and probably play a crucial role in HIV-1 transmission. We investigated the role of cell-to-cell contact between HIV-1-exposed immature DCs and various lymphocyte subsets in the stimulation of HIV-1 replication. We found that HIV-1 replication and production in DCs were substantially enhanced by the coculture of DCs with primary CD4 T or nonpermissive B lymphocytes but not with primary activated CD8 T lymphocytes or human transformed CD4 T lymphocytes. Most of the new virions released by cocultures of HIV-1-exposed immature DCs and primary B lymphocytes expressed the DC-specific marker CD1a and were infectious for both immature DCs and peripheral blood mononuclear cells (PBMCs). Cocultured DCs thus produced large numbers of infectious viral particles under these experimental conditions. The soluble factors present in the supernatants of the cocultures were not sufficient to enhance HIV-1 replication in DCs, for which cell-to-cell contact was required. The neutralizing monoclonal antibody IgG1b12 and polyclonal anti-HIV-1 sera efficiently blocked HIV-1 transfer to CD4 T lymphocytes but did not prevent the increase in viral replication in DCs. Neutralizing antibodies thus proved to be more efficient at blocking HIV-1 transfer than previously thought. Our findings show that HIV-1 exploits DC-lymphocyte cross talk to upregulate replication within the DC reservoir. We provide evidence for a novel mechanism that may facilitate HIV-1 replication and transmission. This mechanism may favor HIV-1 pathogenesis, immune evasion, and persistence.

Non-neutralizing Antibodies Targeting the V1V2 Domain of HIV Exhibit Strong Antibody-Dependent Cell-mediated Cytotoxic Activity

The development of an effective vaccine against HIV-1 has proven to be challenging. Broadly neutralizing antibodies (bNAbs), whilst exhibiting neutralization breadth and potency, are elicited only in a small subset of infected individuals and have yet to be induced by vaccination. Case-control studies of RV144 identified an inverse correlation of HIV-1 infection risk with antibodies (Abs) to the V1V2 region of gp120 with high antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing activity of Abs was not found to contribute to this protective outcome. Using primary effector and target cells and primary virus isolates, we studied the ADCC profile of different monoclonal Abs targeting the V1V2 loop of gp120 that had low or no neutralizing activity. We compared their ADCC activity to some bNAbs targeting different regions of gp120. We found that mAbs targeting the V1V2 domain induce up to 60% NK cell mediated lysis of HIV-1 infected PBMCs in a physiologically relevant ADCC model, highlighting the interest in inducing such Abs in future HIV vaccine trials. Our data also suggest that in addition to neutralization, lysis of infected cells by Abs can effectively participate in HIV protection, as suggested by the RV144 immune correlate analysis.

Neutralizing antibodies inhibit HIV-1 infection of plasmacytoid dendritic cells by an FcγRIIa independent mechanism and do not diminish cytokines production.

Plasmacytoid dendritic cells (pDC) expressing FcγRIIa are antigen-presenting cells able to link innate and adaptive immunity and producing various cytokines and chemokines. Although highly restricted, they are able to replicate HIV-1. We determined the activity of anti-HIV-1 neutralizing antibodies (NAb) and non-neutralizing inhibitory antibodies (NNIAb) on the infection of primary pDC by HIV-1 primary isolates and analyzed cytokines and chemokines production. Neutralization assay was performed with primary pDC in the presence of serial antibodies (Ab) concentrations. In parallel, we measured the release of cytokines and chemokines by ELISA and CBA Flex assay. We found that NAb, but not NNIAb, inhibit HIV-1 replication in pDC. This inhibitory activity was lower than that detected for myeloid dendritic cells (mDC) infection and independent of FcγRIIa expressed on pDC. Despite the complete protection, IFN-α production was detected in the supernatant of pDC treated with NAb VRC01, 4E10, PGT121, 10-1074, 10E8, or polyclonal IgG44 but not with NAb b12. Production of MIP-1α, MIP-1β, IL-6, and TNF-α by pDC was also maintained in the presence of 4E10, b12 and VRC01. These findings suggest that pDC can be protected from HIV-1 infection by both NAb and IFN-α release triggered by the innate immune response during infection.

Broadly Neutralizing Antibody VRC01 Prevents HIV-1 Transmission from Plasmacytoid Dendritic Cells to CD4 T Lymphocytes

ABSTRACT Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-α) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-α secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging

Abstract Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

Translational regulation of APOBEC3G mRNA by Vif requires its 5′UTR and contributes to restoring HIV-1 infectivity

The essential HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells expressing cytidine deaminases APOBEC3G (A3G) and A3F by decreasing their cellular level, and preventing their incorporation into virions. Unlike the Vif-induced degradation of A3G, the functional role of the inhibition of A3G translation by Vif remained unclear. Here, we show that two stem-loop structures within the 5′-untranslated region of A3G mRNA are crucial for translation inhibition by Vif in cells, and most Vif alleles neutralize A3G translation efficiently. Interestingly, K26R mutation in Vif abolishes degradation of A3G by the proteasome but has no effect at the translational level, indicating these two pathways are independent. These two mechanisms, proteasomal degradation and translational inhibition, similarly contribute to decrease the cellular level of A3G by Vif and to prevent its incorporation into virions. Importantly, inhibition of A3G translation is sufficient to partially restore viral infectivity in the absence of proteosomal degradation. These findings demonstrate that HIV-1 has evolved redundant mechanisms to specifically inhibit the potent antiviral activity of A3G.